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A 10-year retrospective comparison of two target sequences, REP-529 and B1, for Toxoplasma gondii
detection by quantitative PCR
Sorya Belaz, Jean-Pierre Gangneux, Peggy Dupretz, Claude Guiguen, Florence Robert-Gangneux
To cite this version:
Sorya Belaz, Jean-Pierre Gangneux, Peggy Dupretz, Claude Guiguen, Florence Robert-Gangneux. A 10-year retrospective comparison of two target sequences, REP-529 and B1, for Toxoplasma gondii detection by quantitative PCR. Journal of Clinical Microbiology, American Society for Microbiology, 2015, 53 (4), pp.1294-1300. �10.1128/JCM.02900-14�. �hal-01118293�
1
A 10-year retrospective comparison of two target sequences, REP-529 and B1, for Toxoplasma 1
gondii detection by quantitative PCR 2
3
Running title: REP-529 in clinical diagnosis 4
5
Sorya BELAZ 1,2, Jean-Pierre GANGNEUX 1,2, Peggy DUPRETZ 2, Claude GUIGUEN 2, Florence ROBERT- 6
GANGNEUX 1,2 7
8
1 INSERM U1085, Institut de Recherche en Santé Environnement et Travail, Université Rennes 1, 9
Rennes, France.
10
2 Laboratoire de Parasitologie et Mycologie, Centre Hospitalier Universitaire de Rennes, Rennes, 11
France.
12 13
Corresponding author:
14
Florence Robert-Gangneux, Centre hospitalier et universitaire de Rennes, 2 rue Henri le Guilloux, 15
35033 Rennes Cedex, France 16
Florence.robert-gangneux@univ-rennes1.fr 17
18
Key-points (40 words): Few studies analyzed the impact of the lower sensitivity of B1-based 19
quantitative PCR (qPCR) for the molecular diagnosis of toxoplasmosis. Our results show that B1 qPCR 20
amplified only 54.4% of REP-529-positive clinical samples prospectively obtained from patients with 21
toxoplasmosis.
22 23 24
Key words: diagnosis, toxoplasmosis, qPCR, Toxoplasma B1, Toxoplasma REP-529 25
26
2 Abstract
27 28
This study aimed to evaluate the repeated sequence REP-529 compared to the B1 gene for the 29
molecular diagnosis of toxoplasmosis by quantitative PCR (qPCR) in routine diagnosis.
30
Over a ten-year period (2003-2013), all patients prospectively diagnosed with a positive REP-529 31
qPCR result for toxoplasmosis were included. All DNA samples (76 samples from 56 patients) were 32
simultaneously tested using the two qPCR methods (REP-529 and B1).
33
The mean Ct obtained with the B1 qPCR was significantly higher (+4.71 cycles) than that obtained 34
with REP-529 qPCR (p<0.0001). Thirty-one out of 69 extracts (45.6%) positive with REP-529 qPCR 35
were not amplified with the B1 qPCR (relative sensitivity 54.4%, compared to REP-529), yielding false 36
negative results on 15/28 placentas, 5 cord blood, 2 amniotic fluids, 4 cerebrospinal fluids, 1 aqueous 37
humor, 2 lymph node punctures and 1 abortion product. This defect in sensitivity would have left 38
20/56 patients undiagnosed, distributed as follows: 12/40 congenital toxoplasmosis, 4/5 cerebral 39
toxoplasmosis, 2/8 patients with retinochoroiditis, and both patients with chronic lymphadenopathy.
40
This poor performance of B1 qPCR could be related to low parasite loads, since the mean 41
Toxoplasma quantification in extracts with B1 false negative results was 0.4 parasite/reaction.
42
These results clearly show the superiority of the REP-529 sequence in the diagnosis of toxoplasmosis 43
by PCR suggest that this target should be adopted as part of standardization of the PCR assay.
44 45 46
3 Introduction
47 48
Toxoplasmosis is a worldwide parasitic infection due to the intracellular parasite Toxoplasma gondii.
49
The infection is usually asymptomatic in immunocompetent patients, and more rarely result in fever, 50
lymphadenopathy or retinochoroiditis. By contrast, immunocompromised patients can experiment 51
severe neurologic, ocular, pulmonary or disseminated disease (1). Yet toxoplasmosis is well-known 52
for its pathogenicity during pregnancy. Indeed, when primary infection occurs in pregnant women, it 53
can lead to congenital toxoplasmosis, with a frequency of transmission and a severity of fetal 54
infection depending on the stage of pregnancy at which infection occurs (2). Diagnosis of 55
toxoplasmosis is routinely based on serology. In some countries, such as France, seronegative 56
pregnant women are monitored monthly by serology. In case of seroconversion, detection of 57
Toxoplasma gondii DNA by PCR is a major diagnostic method for congenital toxoplasmosis, and is 58
performed on amniotic fluid (prenatal diagnosis)(3-5), and placenta or cord blood at birth (postnatal 59
diagnosis)(6-8). In immunocompromised patients, DNA can also be found in cerebro-spinal fluid 60
(CSF), broncho-alveolar lavage (BAL) or others samples guided by clinical signs. The 35-fold repeated 61
B1 gene (9) has been commonly used for this molecular diagnosis since 1989 with acceptable 62
sensitivity (3, 5, 10), but another sequence (REP-529, Genbank AF146527) was described more 63
recently, as being 200- to 300-fold repeated (11), leading to better detection of low parasite loads 64
using spiked specimens (12). Our objective was to evaluate the diagnostic gain resulting from the 65
routine use of a quantitative PCR (qPCR) targeting REP-529, compared to a qPCR targeting the B1 66
gene, for the diagnosis of toxoplasmosis in various clinical settings. In this study, we analyzed 67
retrospectively the qPCRs results of all T. gondii-positive DNA samples from patients who benefited 68
from molecular diagnosis over a 10-year period.
69 70
Materials and methods 71
72
Patients and sample collection 73
During the study period (2003-2013), all patients with a diagnosis of congenital toxoplasmosis who 74
benefited from a molecular diagnosis on at least one sample (amniotic fluid, placenta or cord blood) 75
were included. Congenital toxoplasmosis was defined by a positive prenatal diagnosis for 76
Toxoplasma gondii (molecular diagnosis and/or mouse inoculation) and/or serologic evidence of 77
antibody synthesis by the newborn at birth or during the one-year serologic monitoring (specific IgM 78
or IgA detected by ISAGA (BioMérieux, Marcy-l’Etoile, France) or neosynthesized IgG detected by 79
western-blot (LD-Bio, Lyon, France)). During the study period, routine qPCR molecular diagnosis 80
4
relied on both gene targets (B1 and REP-529) from 2003-2005, then only REP-529 has been used until 81
now. All positive samples were re-analyzed with both gene targets in the same qPCR run, to 82
homogenize parasite quantification, as standard curve had changed meanwhile. Additionally, all 83
samples from proven congenitally infected infants which had been tested negative in routine 84
diagnosis with REP-529 were also retested in parallel with the B1 PCR.
85
Besides, adult patients with clinical signs compatible with toxoplasmosis (lymph nodes with specific 86
anti-Toxoplasma IgM, uveitis, retinochoroiditis, cerebral abscess) and a positive DNA detection with 87
REP-529 from 2009-2013 were also included, and DNA samples were re-analyzed with both qPCRs.
88 89
Molecular diagnosis 90
On reception of the placenta (PL), several samples were taken from different sites and mixed in a 91
solution containing 2.5 mg/mL trypsin, penicillin 500 IU/mL, and gentamicin 3.3µg/mL. The 92
preparation was digested for 2 hours at 37°C under agitation, filtered through gauze and centrifuged 93
for 10 minutes at 1000 g. The supernatant was discarded and the pellet was washed 3 times. Then, 94
200µl of prepared placentas and other crushed biopsies (lymph-node or cerebral) were digested 95
overnight with proteinase K at 56°C prior to extraction. Amniotic fluids (AF) samples (10mL) were 96
centrifuged at 1500xg for 10 min then supernatant was discarded and 2x200µL samples of the pellet 97
were used for DNA extraction. Other fluids (CSF, aqueous humor (AH)) were centrifuged at 1500xg 98
for 10 min, and 200µL of pellet was used for DNA extraction. Routinely, DNA extraction of all samples 99
was performed using QIAmp DNA mini kit® columns (Qiagen, Courtaboeuf, France) and eluted in 100
100µL, except for blood (peripheral blood (PB) and cord blood (CB)) samples, which were processed 101
using 1-2 mL of whole blood and extracted using QIAamp DNA midi kit® columns (Qiagen), and 102
eluted in 400 µL. Two extracts were performed for amniotic fluids and placentas (only one for other 103
samples) and amplification was run in duplicate for all samples.
104
The primers and probe used to amplify a 98 pb fragment of the B1 gene were 5’-GAA AGC CAT GAG 105
GCA CTC CA-3’ (forward) and 5’ –TTC ACC CGG ACC GTT TAG C- 3’ (reverse), and FAM™-5’-CGG GCG 106
AGT AGC ACC TGA GGA GAT ACA-3’-TAMRA™ (13), and primers and probe targeting REP-529 were 107
those described previously (6). All samples were re-analyzed by both qPCRs in the same run, using 108
the following conditions: 25µl reaction mixture containing primers and probe at a final concentration 109
of 600 nM and 200 nM, respectively, 12.5 µL of TaqMan™ Universal PCR Master mix (Applied 110
Biosystems, Courtaboeuf, France), and 5 µL of DNA sample. Amplification was performed on a Step 111
One plus device (Applied Biosystems) for 40 cycles (15 sec 95°C and 1 min 60°C) preceded by 10 min 112
at 55°C for UNG reaction and 10 min denaturation at 95°C. Appropriate controls were included in 113
each run (positive and negative controls, internal control of inhibition). Positive samples were 114
5
quantified using standard curve obtained by serial dilutions of a standardized control (105 115
Toxoplasma RH strain), provided by the National Reference Center for Toxoplasmosis. In the aim to 116
compare quantification data over the study period, all positive samples were re-analyzed for 117
quantification using the same standard curve. The cycle threshold (Ct) of positive samples was 118
recorded for comparison of REP-529 and B1 qPCRs. All Ct results previously acquired during routine 119
analysis and results acquired after retesting for parasite quantification for the purpose of this study 120
were compared to ensure that long storage did not alter DNA.
121 122
Statistical analysis 123
Before comparing the sensitivity of REP-529 and B1 targets, we verified the quality of DNA samples 124
stored at -20°C, by using a Wilcoxon paired test to compare the Ct newly obtained with REP-529 125
qPCR to the Ct previously recorded in routine diagnosis.
126
For the statistical analysis of relative sensitivity between B1 and REP-529 qPCRs, paired Ct obtained 127
with both qPCRs were compared using the Wilcoxon test. The mean Ct obtained with B1 or REP-529 128
qPCR per type of sample were also compared using the Mann-Whitney test. Only newly acquired 129
quantitative results obtained with both PCR simultaneously were taken into account for statistical 130
analysis. Absolute sensitivity of each PCR for the diagnosis of congenital toxoplasmosis was evaluated 131
on the whole cohort of congenitally infected infants diagnosed over the study period who benefited 132
either from prenatal diagnosis or neonatal diagnosis by PCR.
133
Statistical analysis was made using GraphPad® Prism V5 (GraphPad software, USA). P < 0.05 was 134
considered significant.
135 136
Results 137
138
Patients and samples 139
Forty-one cases of congenital toxoplasmosis were included, consisting of 20 amniotic fluids, 1 140
abortion product, 35 placentas and 5 cord blood samples. Additionally, 7 cases of reactivation 141
toxoplasmosis in immunocompromised patients (2 blood samples, 4 CSF, 1 vitreous fluid and 1 142
cerebral biopsy) and 7 cases of symptomatic toxoplasmosis in immunocompetent patients were 143
included (2 lymph node biopsy specimens, 5 aqueous humors). Overall, 76 samples from 56 patients 144
were analyzed. Clinical and biological data of the patients are detailed in Table 1.
145 146
Lack of impact of storage on qPCR Ct 147
6
Forty-nine samples were retrospectively re-analyzed with B1 qPCR in this work, thus it was essential 148
to verify that long-term storage at -20°C after initial diagnosis had not altered DNA. The mean Ct of 149
REP-529 qPCR obtained on samples before (at time of diagnosis) and after storage were similar (31.9 150
± 0.8 versus 32.1 ± 0.8, p=0.8347). Additionally, the Ct results were compared using a Wilcoxon 151
paired test, which confirmed that they did not differ (p = 0.1631, data not shown), thus making 152
possible the interpretation of the data.
153 154
Comparison of B1 qPCR vs REP529 qPCR 155
The performance of both qPCR targets was analyzed on 76 samples. Overall, 31 of 69 extracts (45%) 156
tested positive with REP-529 qPCR were not amplified by the B1 qPCR, thus the relative sensitivity of 157
the B1 qPCR was 55%, compared to the REP-529 qPCR (Table 2). Seven false negative results (7 158
placenta samples) were also observed with REP-529; none was positive with the B1 qPCR. Thus, over 159
the study period, the absolute sensitivity of REP-529 qPCR and B1 qPCR on placenta samples was 160
80% and 37%, respectively (Tables 1&3). Their absolute specificity for prenatal diagnosis was 100%
161
and 90%, respectively (Table 3). False negative results with the B1 qPCR, compared to REP-529 qPCR, 162
were observed on all sample types, yet mainly on placenta samples (15/28, 54%), blood samples 163
(6/7, 86%), and CSF (4/4, 100%) (Table 2). Overall, the mean Ct obtained with REP-529 qPCR when B1 164
qPCR was negative was 36.84 ± 0.36 (corresponding to 0.37 ± 0.3 parasites/reaction), underlining the 165
need for a very sensitive qPCR assay. For the 38 samples which could be amplified with both qPCRs, 166
the mean Ct obtained with the B1 qPCR was significantly higher (+4.7 ± 0.3 Ct) than that obtained 167
with REP-529 qPCR (p<0.001) (Table 4). The mean gain in amplification Ct ranged from 3.65 to 4.98 168
according to the type of sample, and was highly significant for amniotic fluids and placenta samples 169
(p<0.001, Table 4). The difference was not statistically significant for aqueous humor samples, 170
probably because of a lack of statistical power.
171
Regarding congenital toxoplasmosis, the B1 qPCR failed to amplify any of the available samples (AF, 172
PL or CB) in 11 out of 40 cases (27.5%) which were positive with REP-529 qPCR. This defect in 173
sensitivity would have had variable consequences according to the results of other biological 174
techniques used. Importantly, prenatal diagnosis would have been falsely negative in two cases (#19 175
and 31), and fetal infection would have remained unproven in a case of fetal loss (#17). Of note, B1 176
target also yielded a false negative result on the placenta sample from case #19 (Table 1). In three 177
other cases (#20, 26, 27), where no prenatal diagnosis had been performed because of late maternal 178
infection during the third trimester of pregnancy, the B1 qPCR would have left the newborns 179
undiagnosed, since no serological evidence of infection was observed until several months (Table 1).
180
For the five remaining cases (#8, 11, 15, 22, 30), the consequences of the reduced sensitivity of B1 181
7
qPCR would have been negligible, as serological markers of congenital infection (specific IgM or IgA 182
detection, neosynthesized IgG on western-blots) were observed in newborns at birth or during the 183
first week of live. In case #15, B1 qPCR was falsely negative in both placenta and cord blood. In the 184
remaining cases, the lack of sensitivity of B1 on placenta samples was moot, since the diagnosis had 185
been made previously on AF.
186
Regarding ocular toxoplasmosis, one aqueous humor from an immunocompetent patient for whom 187
Western blot was not contributive, and one blood sample from a HIV+ patient, who did not undergo 188
aqueous humor puncture (Table 1), were negative with B1, which would have left 2 patients 189
undiagnosed. Additionally, the B1 target would have left undiagnosed 4 out of 5 (80%) cerebral 190
toxoplasmosis, and the two patients with chronic lymphadenopathy (Table 2).
191 192
Discussion 193
194
This study including all positive samples obtained by REP-529 qPCR over a 10-year period, clearly 195
shows the superiority of the REP-529 target for the diagnosis of toxoplasmosis, whatever the clinical 196
setting. As previously described in other studies, this can be explained by the difference in the 197
number of repetitions of this sequence (about 7- to 10 fold more repeated than B1)(11), which 198
results in a gain of about 4 Ct (12, 14), as observed here (p<0.001), and allows detection of 10-fold 199
lower parasite loads (14). However, few studies evaluated the impact in terms of sensitivity of 200
diagnosis in routine clinical use.
201
Filisetti et al. (15) compared three PCR methods on 23 selected AF samples and 16 CB from 19 cases 202
of congenital toxoplasmosis, and found that only 5 out of 9 REP-529-positive AF were positive with 203
the B1 PCR. It must be noted that the overall sensitivity in this study was very low, since only 11 out 204
of 19 AF (58%) were positive with the reference method used, i.e. rDNA PCR (16), which could be due 205
to the use of conventional PCR methods in this study. Cassaing et al. (14) included 33 positives 206
samples (8 AF, 15 PL, 3 AH, 3 CSF, 2 blood samples and 2 BAL), of which 13/15 (87%) PL and 8/8 AF 207
(100%) tested positive with REP-529 qPCR were also positive with B1 qPCR. No differences in 208
sensitivity between both targets were observed for AH, CSF and BAL samples, but all samples were 209
tested in duplicate and in 11 instances, negative results were observed in 1 of the 2 amplifications 210
with B1 qPCR, whereas one sample was amplified by REP-529 qPCR only once. Additionally, the 211
authors considered a B1 result as positive, although it was over 40 Ct, in 4 samples. Another study 212
(17) included prospectively 135 AF samples, of which 27 and 22 were positive with REP-529 and B1 213
qPCR, respectively. The authors declared that 2 of the 5 presumed false negative B1 results were in 214
fact false positive REP-529 results, however, no details are provided on the newborns follow-up and 215
8
the criteria that led them to this conclusion. Finally, the most recent study (18) evaluated three qPCR 216
methods, mainly on AF samples. The authors reported 33 positive results with their B1 qPCR, 217
compared to 43 with two REP-529 qPCR methods, leading to a relative sensitivity of 77% for B1. Two 218
cord blood samples tested positive with REP-529 were negative with B1 qPCR. Additionally, they 219
found that their two REP-529 qPCRs methods and devices (Applied Biosystem and Roche) performed 220
equally.
221
Our study focused on the evaluation of the relative sensitivity of B1 versus REP-529 PCR targets, thus 222
all PCR-positive samples were included. Additionally, all samples (AF, PL) from congenitally infected 223
children with a negative REP-529 qPCR result were retested. No samples with B1-positive and REP- 224
529-negative results were observed, either prospectively or retrospectively, in cases of proven 225
congenital toxoplasmosis. The overall relative sensitivity of the B1 qPCR was only 55%, and its 226
absolute sensitivity was 37% in the setting of congenital toxoplasmosis (Tables 2&3). This defect in 227
sensitivity was particularly crucial for 3 antenatal diagnoses (2 prenatal diagnoses and 1 early fetal 228
loss undiagnosed) and 3 neonatal diagnoses. In case of fetal loss, the recognition of the role of 229
Toxoplasma is important, because it allows to eliminate other causes of spontaneous abortion and 230
avoids useless investigations. On the other hand, the positivity of prenatal diagnosis usually leads to 231
a change in chemotherapy, using pyrimethamine-sulfonamide combination therapy to treat the 232
mother (1-2), which would have been missed in the two B1-negative patients here. Finally, the 233
parasite DNA detection in placenta, even if not yet recognized as a standard criteria for the diagnosis 234
of congenital toxoplasmosis, was shown to have a positive predictive value over 90% in our hands 235
(6), and is at least a strong argument to accurately follow the infant and increase the frequency of 236
serologic testing to confirm infection, with the aim of reducing delay of treatment. We observed here 237
that parasite DNA detection from placenta was the earliest biological sign of infection in neonates 238
with neither IgM nor IgA detection, and recall the interest of this sample in patients for whom 239
antenatal diagnosis was negative or not performed. In 2 cases (#23, 41), both qPCR were negative on 240
placenta samples, whereas they were positive with mouse inoculation, still underlining the interest 241
to combine both techniques (Table 1).
242
The B1 qPCR had also poor sensitivity in other clinical settings. The diagnosis of cerebral 243
toxoplasmosis is difficult, and ancient studies have reported a poor sensitivity of conventional PCR 244
methods (19-20), probably related to low parasite loads, but no recent studies evaluated new qPCR 245
methods in this setting. The present study was not designed to answer this question, but we noted 246
that four patients would have been undiagnosed using B1 qPCR. Besides, the REP-529 qPCR allowed 247
diagnosing two cases of chronic toxoplasmosis in patients with lymphadenopathy and history of 248
9
Toxoplasma seroconversion in the past twelve months, which led to consider this diagnosis and stop 249
further exploration aiming at diagnosing a hematological malignancy.
250
False negative results targeting B1 had a Ct > 34 (mean 36.84 ± 0.36). This poor performance of B1 251
qPCR could be related to low parasite loads, a frequently observed situation in congenital 252
toxoplasmosis, particularly in France where women are treated all along pregnancy, which probably 253
decreases the parasite burden. In the study by Romand et al. (21), 88 positive AF were included, of 254
which 35 (40%) were shown to contain less than 10 parasite/mL.
255
Besides, the proportion of undetected samples with the B1 qPCR was high for placenta samples (57%
256
of REP-529-positive samples) and blood samples (50%). In 12 placenta samples, B1 qPCR was 257
negative whereas mouse inoculation was positive, which is unusual. Poor sensitivity on blood and 258
placenta samples suggests that inhibitors would more likely interfere with this PCR. Indeed, Chabbert 259
et al. (22) nicely showed that the efficacy of PCR on samples spiked with low amounts of parasites 260
was lower in placenta or blood, compared to AF, with both sets of B1 primers used.
261
After the description of the REP-529 sequence, the B1 target has been still frequently used in parallel 262
in most labs, including ours, until it could be demonstrated that the occurrence of B1 positive results 263
and REP-529 negative results was never observed (14-15, 18), suggesting that this sequence is 264
present in all parasite isolates. However, a recent Brazilian study (23) reported a lower proportion of 265
positive amniotic fluids with REP-529 target, than with B1 (36.5% and 87.3%, respectively), with only 266
23.8% of samples being positive with both targets. This unusual finding must be verified on other 267
series of clinical samples from South America, to check if atypical parasite strains circulating in this 268
area could lack the REP-529 sequence or have mutations or modification of the number of 269
repetitions, that could lead to a decreased sensitivity of this PCR target, which now appears to be the 270
gold standard for European parasite strains.
271
Therefore, in view of the results obtained in the present study, we suggest widespread use of the 272
REP-529 qPCR target, which should replace B1 target, at least in Western countries, until its value is 273
confirmed in South America.
274 275
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342 343
12
Table 1: Clinical characteristics and qPCR results for 41 mother-child pairs of congenital toxoplasmosis, 8 cases of ocular toxoplasmosis, 5 cases of cerebral 344
toxoplasmosis and 2 cases of chronic lymphadenopathy 345
346 347
Case N°
Clinical setting
Clinical signs (Pregnancy trimester at maternal infection for CoT)
Sampl e
qPCR results Mouse inoculatio n
Other biological criteria of infection B1 Rep-529
1 CoT Abnormal neurodevelopment (ultrasound) : Medical termination of pregnancy (T1)
AF + + + Positive qPCR (REP) on fetal biopsy
PL + + ND
2 CoT Asymptomatic (T3) AF + + + IgM detection at 3month in the infant (in another
hospital), PL positive by mouse inoculation
3 CoT Asymptomatic (T3) PL + + + Positive prenatal diagnostic in another hospital.
Neosynthesized IgG/IgM by WB; IgM and IgA detection at birth
CB - + ND
4 CoT Asymptomatic (T3) PL + + ND Neosynthesized IgG/IgM by WB; IgM and IgA detection at
1 month
CB - + ND
5 CoT Asymptomatic (T3) PL + + + IgM at birth
6 CoT Asymptomatic (T2) AF + + - Persisting IgG at 1 year of life
PL - + +
7 CoT Ventriculomegaly (ultrasound) : Medical termination of pregnancy (T1)
PL + + + Toxoplasma detection in AF by mouse inoculation
8 CoT Asymptomatic (T3) PL - + + Neosynthesized IgG/IgM by WB; IgM and IgA detection at
birth 9 CoT Intracerebral calcifications
(ultrasound) (T2-3)
AF + + + Persisting IgG at 1 year of life, Toxoplasma detection in PL by mouse inoculation
10 CoT Asymptomatic (T2) PL + + + Neosynthetized IgG by WB
11 CoT Seizure after birth (T3) PL - + + Positive prenatal diagnostic in another hospital,
13
neosynthesized IgM (WB) at birth
12 CoT Unknown (T3) PL + + + Neosynthesized IgG/IgM by WB
13 CoT Intrauterine fetal death (T2) AF + + +
14 CoT Asymptomatic (T3) AF + + + Neosynthesized IgG/IgM, IgM and IgA detection at birth
PL - + +
15 CoT Asymptomatic (T3) PL - + - IgM and IgA detection at 1 week of life
CB - + ND
16 CoT Unknown (T3) PL + + + Neosynthesized IgG/IgM by WB; IgM and IgA detection at
birth
CB - + ND
17 CoT Termination of pregnancy (T1) AP - + ND
18 CoT Chorioretinitis (T2) AF
PL + -
+ -
+ -
IgM detection at birth
19 Co T Asymptomatic (T3) AF - + + Neosynthesized IgG/IgM by WB; IgM and IgA detection at
birth
PL - + +
20 CoT Asymptomatic (T3) PL - + + IgM (in another hospital) at birth
21 CoT Asymptomatic (T3) AF + + + Neosynthesized IgG/IgM by WB at birth
22 CoT Asymptomatic (T3) PL - + + Positive prenatal diagnostic in another hospital
23 CoT Asymptomatic (T2) AF + + + Neosynthesized IgG/IgM by WB; IgM and IgA detection at
birth
PL - - +
24 CoT Chorioretinitis (T2) AF + + + IgM and IgA detection at birth and neosynthesized
IgG/IgM by WB at 1 month
PL - + +
25 CoT Asymptomatic (T1) AF
PL + -
+ -
+ -
26 CoT Unknown (T3) PL - + + Persisting IgG at 1 year of life
27 CoT Asymptomatic (T3) PL - + + Neosynthesized IgG by WB at 3 months of life
28 CoT Chorioretinitis (T3) AF + + + IgM and IgA detection at birth and neosynthesized IgG
(WB) at 1 month of life
PL - + +
29 CoT Asymptomatic (T2) AF + + + Neosynthesized IgG at 3 month (WB)
14
PL - - -
30 CoT Asymptomatic (T3) PL - + - Neosynthesized IgG/IgM by WB; IgM and IgA detection at
birth
31 CoT Asymptomatic (T2) AF - + - DNA detection by REP-529 qPCR in cord blood (B1 not
determined)
PL - - -
32 CoT Asymptomatic (T3) PL + + + Neosynthesized IgG/IgM by WB; IgM and IgA detection at
birth
CB - + ND
33 CoT Asymptomatic (T3) AF + + + IgM and IgA detection at birth, neosynthesized IgG by WB
during follow-up
PL - + +
34 CoT Unknown (T3) PL + + ND Positive prenatal diagnostic in another hospital
35 CoT Chorioretinitis and intracerebral calcifications (T2)
AF PL
+ -
+ -
+ -
Persisting IgG at 1 year of life
36 CoT Asymptomatic (T3) AF + + + Neosynthesized IgG/IgM by WB; IgM and IgA detection at
birth
PL - + -
37 CoT Asymptomatic (T3) PL + + + Neosynthesized IgG/IgM by WB; IgM and IgA detection at
birth
38 CoT Asymptomatic (T3) AF + + +
PL + + +
39 CoT Unknown (T3) AF + + + Positive serological monitoring in another hospital
40 CoT Asymptomatic (T3) PL + + + Positive prenatal diagnostic in another hospital, IgM at
birth
41 CoT Asymptomatic (T3) PL - - + IgM and IgA detection at birth and neosynthesized IgG
(WB)
42 OT Uveitis AH + + ND
43 OT Uveitis AH + + ND Neosynthesized IgG in AH (WB)
44 OT Uveitis and chorioretinitis AH + + ND
45 OT Uveitis AH + + ND Neosynthesized IgG in AH (WB)
46 OT Hyalitis, not treated for toxoplasmosis AH - + ND
15 47 OT Uveitis, HIV + patient. Treated for
toxoplasmosis
VF + + ND Neosynthesized IgG in VF (WB)
48 OT Uveitis, HIV + patient. Treated for toxoplasmosis
PB - + ND Serological reactivation (high levels of IgG and IgM) 49 OT Retinochoroiditis, HIV+ patient.
Treated for toxoplasmosis.
PB + + ND
50 CeT CSF - + ND
51 CeT Neurological symptoms, HIV+ patient CSF - + ND 52 CeT Immunocompromised patient, 5
cerebral lesions on CT scan
CeB + + ND DNA detection in CSF in another hospital 53 CeT Heart transplant patient, neurological
symptoms
CSF - + ND IgM detection in serum
54 CeT Cerebellar syndrome CSF - + ND Serological reactivation (high levels of IgG and IgM) 55 CL Chronic toxoplasmosis, asthenia LNP - + ND History of seroconversion (persisting IgM) 56 CL Chronic toxoplasmosis, asthenia LNP - + ND History of seroconversion 6 month before
CoT, congenital toxoplasmosis; AF, amniotic fluid; PL, placenta; CB, cord blood; AP, abortion product; T1, first trimester of pregnancy; T2, second trimester 348
of pregnancy; T3, third trimester of pregnancy; OT, ocular toxoplasmosis; AH, aqueous humor; PB, peripheral blood; VF, vitreous fluid 349
CeT, cerebral toxoplasmosis; CSF, cerebro-spinal fluid, CeB, cerebral biopsy 350
CL, chronic lymphadenopathy; LNP,lymph node puncture; ND, not determined ; WB, Western-blot 351
352
16
Table 2: Relative sensitivity of B1 qPCR on samples tested positive with REP-529 qPCR, according to 353
the type of sample 354
Sample B1 qPCR result
No positive/Total No (%)
Amniotic fluid 18/20 (90)
Placenta 13/28 (46.4)
Abortion product 0/1 (0)
Cord blood 0/5 (0)
Blood 1/2 (50)
Ocular fluids 5/6 (83)
Cerebrospinal fluid 0/4 (0) Biopsy (cerebral or lymph node) 1/3 (33)
All samples 38/69 (55)
355 356
17
Table 3: Sensitivity of B1 qPCR and REP-529 qPCR for the diagnosis of congenital toxoplasmosis 357
(2003-2013) 358
Sample B1 qPCR result
No positive/Total No (%)
REP-529 qPCR result No positive/Total No (%) Prenatal diagnosis (amniotic fluid) 18/20 (90) 20/20 (100%)
Neonatal diagnosis (placenta) 13/35 (37) 28/35 (80%) 359
360
18
Table 4: Mean Cycle threshold (Ct) obtained with B1 qPCR and REP-529 qPCR, according to sample 361
type.
362 363
Sample Ct B1
(Mean ± SEM)
Ct Rep 529
(Mean ± SEM) (Mean ± SEM)
P valuea P valueb
Amniotic fluid (n = 18) 34.80 ± 0.58 29.87 ± 0.57 4.93 ± 1.35 0.0002*** <0.0001***
Placenta (n = 12) 35.86 ± 0.57 30.87 ± 0.58 4.98 ± 2.07 0.0002*** <0.0001***
Ocular fluids (n =5) 32.98 ± 2.00 29.84 ± 2.30 3.49 ± 1.1 0.0625 0.3095 All samples (n = 37) 34.51 ± 0.56 29.7 ± 0.53 4.81 ± 0.31 <0.0001*** <0.0001
a Wilcoxon test; b Mann Whitney test 364
***, p<0.001 365
366
